Leukemia stem cells (LSCs) are the key subset of leukemic cells (LCs) required for leukemia initiation, progression and relapse. Complete elimination of LSCs is currently the ultimate goal of leukemia therapy. In 2001, it was found that using an inhibitor against NF-?B (a transcriptional regulator of many survival genes) resulted in significant death of LSCs in human acute myeloid leukemia samples while preserving healthy hematopoietic stem cells /progenitors (HSC/Ps).1 Unfortunately, the clinical use of NF-?B inhibitors at therapeutic levels is limited by the development of severe inflammation-related side-effects in the skin, the liver, and elements of the nervous, and immune systems. These side effects were uncovered during testing in mice.2-4. Therefore, we need new therapies that reduce side-effects either by using a better NF-?B inhibitor at a lower dose or by developing novel therapies which prevent the inflammation caused by NF-?B inhibitor. Tumor Necrosis Factor alpha (TNF) is a major inflammatory signal released by hematopoietic cells and is implicated in NF-?B modulation therapies.5, 6 While TNF's inflammatory signals causes cell death and growth arrest in normal HSC/Ps, its role in leukemia development is not yet clear.7, 8 What is clear is that managing inflammation is critical for the successful treatment of leukemia.9, 10 Our preliminary studies show that LCs utilize TNF-mediated inflammation as both a survival and proliferation signal by activating a signaling network that depends in part on NF-?B. By knocking out both receptors of TNF (Tnfr1a-/-1b-/-), we can sensitize LCs 100-fold to NF-?B inhibitor treatment when compared to normal HSC/Ps. Therefore, we hypothesize that inhibiting both TNF receptor signaling and NF-?B would provide a double benefit: clinicians could use NF-?B inhibitors at a lower dose while simultaneously blocking TNF-dependent inflammation. Our broad goal in this study is to identify novel leukemia-specific therapeutic strategies that can augment NF-?B inhibition. Our immediate goal is to determine whether blocking TNF signaling augments NF- ?B inhibition in eliminating both leukemic cells and leukemic stem cells. In our study, we will compare treatments in LCs and bone marrow (BM) HSC/Ps, and will take advantage of the murine genetic leukemia models we have generated. We will utilize pharmacological approaches to alter the activity of signaling pathways in order to maximally kill LCs while preserving normal HSC/Ps. We will measure global effects on cell death by Annexin V/PI staining, colonigenic capacity of progenitor cells by CFU assay, and hematopoietic reconstitutive capacity of normal HSC/Ps and leukemogenic capacity of LSCs by in vivo transplantation. The first aim of our study explores the effects of blocking TNF in conjunction with NF-?B inhibitor treatment. Our second aim explores small molecule inhibition of TNF receptor downstream targets in combination with NF-?B inhibitor treatment. The expected results of this study will significantly contribute to furthering the understanding of leukemia, an may also point the way toward novel strategies for future therapies.